The central goal of this project is to elucidate the mechanism whereby the HIV genome gains entry into the cell. Our overall approach is to kinetically resolve steps in the pathway of HIV Envelope Glycoprotein (Env)-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Resolution of the characteristics of these intermediates will yield insights into the mode of action of the viral envelope glycoproteins and enable us to identify potential antibodies and inhibitors that block HIV entry and prevent infection. We have studied the mode of action of small molecule antagonists of the viral coreceptors CCR5 and CXCR4 and the fusion inhibitor T20 that is in advanced clinical trials. By examining fusion kinetics of various HIV strains we found that the sensitivity of HIV to entry inhibitors correlates with envelope:coreceptor affinity, receptor density and fusion kinetics. An important implication of these findings is that individuals who express lower levels of coreceptor may respond more efficiently to entry inhibitors such as T20, whose effectiveness is dependent in part on fusion kinetics. We found that membrane microdomains enriched in cholesterol and glycosphingolipids are involved in HIV-1 entry into cells in as much as there is a necessity to recruit the fusion complexes in a limited area of the membrane to provide the high surface density of coreceptors. We are pursuing approaches to interfere with HIV-1 entry using agents that change membrane organization. These findings may show a way to interfere with HIV infection by changing cellular levels of ceramides. We have developed new methodologies that include photosensitized labeling, membrane protein purification, tandem proteolysis and Mass Spectroscopy analysis to identify domains of HIV/SIV Env that are involved in the operation of the fusion machine. Information about these domains is not accessible by current high-resolution structural determination techniques. Based on the tools acquired in these studies, we are developing photosensitized labeling as a general methodology to study nearest neighbor interactions between membrane components of signal transduction pathways in normal and transformed cells.